This proposal will focus on the biochemical, molecular and cellular aspects of how hormonal ligands and cofactors influence development and physiology through specific control of gene expression. Understanding the fundamentalprocesses of gene activation by nuclear hormone receptors is a crucial step towards elucidating the biological roles of these receptors in development and homeostasis. The goal of SpecificAim I is to apply a structural approach to examine the basis of ligand-independent interaction of the co-activators PGC-la and 0 with the nuclear hormone receptor PPAR6 by X-ray crystallography. In addition we will also determine through NMR structural studies the functional HAT domain of the nuclear receptor co-activator ACTR. Specific Aim II will use the latest state-of-the-art protein chemistry techniques to isolate and functionally characterize the co-activator complexes of ACTR, PGC-la and PGC10. After isolation each complex and individual component will be tested for enzymatic activity and substrate specificity and well as their importance and role in the protein complex. Specific Aim III will utilize promoter chromatin immunoprecipitation (ChIP) technology to identify gene targets of the co-activators ACTR and PGC-lp in adipocyte and myocyte differentiation.Potential target genes will be examined for their biological significance and confirmed for authenticity through a variety of techniques. Finally in Specific Aim IV we propose to dissect the physiological function of a variety of co-activators using tissue specific co-activator gene knockout mice. Once generated we will examine and measure the metabolic consequences of the gene knockout through the use of metabolic cages. Lipid parameters such as cholesterol, bile acids, fatty acids, insulin and glucose levels will also be measured for differences. Together these experiments will provide us with important new insights into the underlying molecular mechanisms of how nuclear hormone receptors influence broad aspects of endocrine physiology in human homeostasis and disease states.